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Mechanistic insight into the norepinephrine-induced fibrosis in systemic sclerosis

received: 22 January 2016

Akihito Uehara*, Sei-ichiro Motegi*, Kazuya Yamada, Akihiko Uchiyama, Buddhini Perera, Sayaka Toki, Sachiko Ogino, Yoko Yokoyama, Yuko Takeuchi & Osamu Ishikawa

accepted: 05 September 2016 Published: 21 September 2016

Raynaud’s phenomenon is frequently observed in systemic sclerosis (SSc) patients, and cold- or stress-induced norepinephrine (NE) has been speculated to be associated with vasoconstriction. Objective was to elucidate the role of NE in fibrosis in SSc. IL-6 is a potent stimulator of collagen production in fibroblasts. NE enhanced IL-6 production and proliferation more significantly in SSc fibroblasts than in normal fibroblasts. Furthermore, the production of IL-6 and phosphorylation of p38 in SSc fibroblasts was enhanced by adrenergic receptor (AR)β agonist, isoproterenol, but not ARα agonist, oxymetazoline. ARβ blocker, propranolol, inhibited NE-induced IL-6 production and phosphorylation of p38 in SSc fibroblasts. NE-induced IL-6 was significantly inhibited by p38 inhibitor, SB203580, suggesting that NE-induced phosphorylation of p38 via ARβ enhances IL-6 production in SSc fibroblasts. NE-induced phosphorylation of ERK1/2 via ARα inhibited IL-6 production in SSc fibroblasts. Combined treatment with NE and endothelin-1 resulted in an additive increase in IL-6 production in SSc fibroblasts. NE-induced IL-6/IL-6 receptor trans-signaling increased the production of collagen type I in SSc fibroblasts, and both propranolol and SB203580 inhibited NE-induced collagen production. These results suggest that cold exposure and/or emotional stress-induced NE might contribute to the skin fibrosis via potentiation of IL-6 production from fibroblasts in SSc. Norepinephrine (NE) is primarily released from the postganglionic neurons of the sympathetic nervous system as a neurotransmitter. NE is also synthesized in the locus coeruleus and adrenal medulla. It is well known that emotional stress and cold stimulation increase the systemic and/or local NE levels1–3. NE binds to the adrenergic receptor (AR) in multiple organs, including the heart, lungs, brain and skin. In the skin, ARβ​2 is primarily expressed on the surface of keratinocytes, dermal fibroblasts and melanocytes4–6. Activation of ARβ​2 signaling impairs re-epithelialization, resulting in delayed wound healing in both human and murine skin7,8. In in vitro studies, activation of ARβ​2 by isoproterenol enhances migration and alters both the actin cytoskeleton and focal adhesion distribution in dermal fibroblasts, suggesting that NE:AR signaling regulates the function of dermal fibroblasts7–9. Systemic sclerosis (SSc) is a connective tissue disorder characterized by the development of fibrosis in the skin and internal organs as well as microvascular dysfunction. Raynaud’s phenomenon is commonly observed in patients with SSc and characterized by the presence of episodic vasospasms and ischemia of the extremities in response to cold or emotional stress. It has also been speculated that cold- or stress-induced NE stimulates ARα​ on pericytes and/or vascular smooth muscle cells, thus resulting in vasoconstriction10,11. In addition, SSc patients treated with the ARα​2c antagonist exhibit improvements in the symptoms of Raynaud’s phenomenon induced by cold stimulation12, suggesting that NE is involved in the pathogenesis of vasculopathy in SSc. However, the roles of NE in the development of skin fibrosis associated with SSc are not well investigated. IL-6 is a pleiotropic multifunctional cytokine produced by various cells, such as lymphocytes, monocytes and fibroblasts13. IL-6 has various immunological functions, for example, it induces B cell differentiation to produce immunoglobulin, stimulates Th17 differentiation in the presence of transforming growth factor (TGF)-β​ and inhibits the induction of TGFβ​-induced regulatory T cells14,15. In addition, IL-6 is considered to be involved in the pathogenesis of several autoimmune diseases, including SSc and rheumatoid arthritis16. With respect to IL-6 and SSc, many in vivo and in vitro studies have shown that IL-6 plays an important role in the pathogenesis of Department of Dermatology, Gunma University Graduate School of Medicine, Japan. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.-i.M. (email: smotegi@ gunma-u.ac.jp)

Scientific Reports | 6:34012 | DOI: 10.1038/srep34012

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www.nature.com/scientificreports/ fibrosis in SSc. For example, the serum IL-6 levels are significantly elevated in SSc patients of early stage17,18 and correlate with the total skin thickness score in persons with this disease19. Moreover, a prominent expression of IL-6 is observed in dermal fibroblasts, mononuclear cells and endothelial cells in the patient’s skin of early stage of diffuse cutaneous type (dc)SSc18,20, suggesting that dermal fibroblasts are an important source of IL-6 in affected skin lesions. In in vitro studies, skin dermal fibroblasts derived from SSc patients have been found to produce high levels of IL-6, and the complex of IL-6 and soluble IL-6 receptor (sIL-6R) has been shown to stimulate SSc fibroblasts via gp130 to differentiate and proliferate, resulting in collagen overproduction and fibrosis18,21–23. In addition, several case studies have reported softening of the skin in SSc patients after the treatment with an anti-IL-6 receptor antibody (tocilizumab), supporting the essential role of IL-6 in the pathogenesis of skin fibrosis associated with SSc24. Recently, phase II clinical trial on tocilizumab in SSc patients revealed that the reduction of skin sclerosis in tocilizumab group tended to be greater than those in placebo group25. It has also been reported that IL-1α​, platelet-derived growth factor (PDGF), tumor necrosis factor-α​ (TNF-α​) and CD154/ CD40 interactions induce IL-6 production in fibroblasts22,26–28. However, the precise mechanisms underlying the onset of IL-6-induced fibrosis in the setting of SSc and whether NE stimulation enhances IL-6 production in SSc fibroblasts remain unclear. In addition, the relationship between NE and endothelin-1 (ET-1), which is associated with skin fibrosis in SSc patients, in the pathogenesis of skin sclerosis of SSc is unknown. In this study, we examined the mechanisms of NE-induced IL-6 production in SSc fibroblasts and aimed to clarify the roles of NE in the pathogenesis of fibrosis in SSc.

Results

NE-induced IL-6 production was significantly higher in the SSc fibroblasts than in the normal fibroblasts.  In order to assess the effects of NE on IL-6 production from normal and SSc dermal fibroblasts,

the secreted protein and mRNA levels of IL-6 in normal and SSc fibroblasts treated with NE were examined. The mRNA and secreted protein levels of IL-6 in the normal and SSc fibroblasts were increased by NE stimulation for one hour in a dose-dependent manner (Fig. 1A,B). Furthermore, the IL-6 mRNA and secreted protein levels in the SSc fibroblasts treated with NE were significantly higher than those observed in the normal fibroblasts treated with NE (Fig. 1A,B). Next, we analyzed the IL-6 mRNA and secreted protein levels in the normal and SSc fibroblasts treated with 10 μ​M of NE for the indicated time. The IL-6 mRNA levels in the normal and SSc fibroblasts increased after NE stimulation, peaking at one hour of stimulation, and then decreased to the basal values at three hours after stimulation (Fig. 1C). In addition, the IL-6 mRNA levels in the SSc fibroblasts treated with 10 μ​M of NE for one hour were significantly higher than those in the normal fibroblasts (Fig. 1C). The secreted protein levels of IL-6 from the normal and SSc fibroblasts were increased by NE treatment in a time-dependent manner (Fig. 1D), and the IL-6 secreted protein levels from the SSc fibroblasts treated with 10 μ​M of NE for six hours were significantly higher than those in the normal fibroblasts (Fig. 1D). These results suggest that NE enhances IL-6 production in normal and SSc fibroblasts in a dose- and time-dependent manner and that the NE-induced IL-6 production is significantly higher in SSc fibroblasts than in normal fibroblasts.

NE-induced IL-6 production in both the normal and SSc fibroblasts was mediated primarily via ARβ.  NE binds both ARα​and ARβ​to activate the downstream signaling. However, NE has different mech-

anisms of activation for ARα​and ARβ​, depending on the specific G protein subunits activated29,30. In order to examine whether the NE-induced IL-6 expression is conducted via ARα​or ARβ​, we assessed the IL-6 mRNA and secreted protein levels in normal and SSc fibroblasts treated with oxymetazoline (ARα​agonist) or isoproterenol (ARβ​agonist) for one hour. While oxymetazoline did not affect the IL-6 mRNA and secreted protein levels in the normal or SSc fibroblasts (Fig. 1E,F), isoproterenol elevated the IL-6 mRNA and secreted protein levels in both the normal and SSc fibroblasts (Fig. 1G,H). Next, we examined the effects of the ARβ​inhibitor, propranolol, on NE-induced IL-6 production in normal and SSc fibroblasts. Consequently, propranolol significantly inhibited the NE-induced IL-6 mRNA and secreted protein levels in the SSc fibroblasts (Fig. 1I,J). These results suggest that the NE-induced IL-6 production in normal and SSc fibroblasts is mediated primarily via ARβ​.

Expression of ARα and ARβ in the normal and SSc fibroblasts.  Next, we explored the expression of AR in normal and SSc fibroblasts treated with or without NE. Consequently, NE did not affect the ARα​1B, ARα​1D or ARβ​1 expression levels in the normal or SSc fibroblasts (Supplemental Figure S1A–C). There was a tendency for the ARβ​2 expression to be higher in the normal and SSc fibroblasts treated with NE than in those treated without NE, however, there were no significant differences (Supplemental Figure S1D). In addition, there was a tendency for the ARα​1 expression to be higher in SSc fibroblasts than in normal fibroblasts (Supplemental Figure S1A,B). However, there were no significant differences in the surface protein expression levels of ARα​1 between the normal and SSc fibroblasts by immunofluorescence staining (Supplemental Figure S1E). NE-induced phosphorylation of p38 via ARβ enhanced the IL-6 production in the SSc fibroblasts.  It has been reported that stimulation of ARβ​enhances the phosphorylation of p38 in cardiac fibro-

blasts31. In addition, p38 is known to be involved in the production of IL-6 in synovial and gingival fibroblasts32,33. These findings suggest that p38 signaling is associated with NE-induced IL-6 production in human dermal fibroblasts. In order to assess this hypothesis, we examined the phosphorylation of p38 in normal and SSc fibroblasts treated with NE, oxymetazoline or isoproterenol. The phosphorylation of p38 in the normal and SSc fibroblasts was significantly enhanced by the ARβ​ agonist, isoproterenol, but not the ARα​agonist, oxymetazoline (Fig. 2A,C). In addition, the phosphorylation of p38 in the normal and SSc fibroblasts was significantly enhanced by NE, and the NE-induced p38 phosphorylation was inhibited by the ARβ​inhibitor, propranolol (Fig. 2B,D). These results suggest that NE-induced p38 phosphorylation is primarily mediated via ARβ​. Furthermore, the NE-induced IL-6 mRNA and secreted protein levels in the Scientific Reports | 6:34012 | DOI: 10.1038/srep34012

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Figure 1.  NE-induced IL-6 production via ARβ and NE-induced proliferation in SSc fibroblasts were significantly higher than those in normal fibroblasts. (A,B) Effects of NE on IL-6 mRNA (A) and IL-6 protein (B) secreted into the media from normal and SSc fibroblasts. Normal and SSc fibroblasts were incubated during 1 hour with indicated concentration of NE. (C, D) Effects of NE on IL-6 mRNA (C) and IL-6 protein (D) secreted into the media from normal and SSc fibroblasts. Normal and SSc fibroblasts were incubated with 10 μ​M NE for indicated time. (E, F) IL-6 mRNA (E) and secreted protein (F) into the media in normal and SSc fibroblasts treated with oxymetazoline (Ox: ARα​agonist) for 1 hour. (G,H) IL-6 mRNA (G) and secreted protein (H) into the media in normal and SSc fibroblasts treated with isoproterenol (Iso: ARβ​agonist) for 1 hour. (I,J) IL-6 mRNA (I) and secreted protein (J) into the media in normal and SSc fibroblasts treated with NE (10 μ​M) and/or ARβ​inhibitor, propranolol (Ppl) for 1 hour. n =​  3-6 samples. mRNA levels in normal fibroblasts without treatments were assigned a value of 1. All values represent mean ±​  SEM. **P